1. Field of the Invention
The invention relates to cyanide-tolerant nitrile hydratases especially from Pseudomonas putida or Pseudomonas marginalis strains which exhibit increased cyanide tolerance, to their use for preparing amides from nitrites in the presence of cyanides and to polynucleotide sequences coding for this enzyme.
2. Description of the Related Art
The conversion of α-hydroxy nitrites (cyanohydrins) and α-amino nitrites into the corresponding amides using nitrile hydratases opens up a novel variant for synthesizing α-hydroxy acids and α-amino acids because α-hydroxy amides and α-amino amides can be hydrolyzed in a simple manner (Process and catalysts for the production of methionine. Ponceblanc, Herve; Rossi, Jean-Christophe; Laval, Philip; Gros, Georges. (Rhone-Poulenc Animal Nutrition SA, Fr.), (WO 2001060789). Alternatively, α-hydroxy amides can also be reacted with alkali metal or alkaline earth metal hydroxides to give the corresponding salts of the hydroxy acids. A particularly preferred reaction in this connection is that of 4-methylthio-α-hydroxybutyramide (MHA amide) with calcium hydroxide, because calcium MHA can be employed directly as alternative form of product to methionine or MHA as feed additive.
However, α-hydroxy nitrites and α-amino nitrites readily decompose to aldehydes and hydrocyanic acid, and aldehydes, hydrocyanic acid and ammonia, respectively. The resulting hydrocyanic acid is a strong inhibitor of almost all known nitrile hydratases with the exception of the nitrile hydratase from Rhodococcus equi XL-1, which shows the smallest loss of activity known to date at 20 mM cyanide (Production of amides from nitrites by Rhodococcus equi cells having a cyanide resistant-nitrile hydratase. Nagasawa, Tohru; Matsuyama, Akinobu. (Daicel Chemical Industries, Ltd., Japan), (EP 1 266 962 A).
The low productivity of about 8 g of amide per g of dry biomass of resting cells, the long reaction time of 43 hours and the relatively low product concentration of 75 g/l lead to the search for improved nitrile hydratases.